CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production

A M Thornton, E M Shevach, A M Thornton, E M Shevach

Abstract

Peripheral tolerance may be maintained by a population of regulatory/suppressor T cells that prevent the activation of autoreactive T cells recognizing tissue-specific antigens. We have previously shown that CD4+CD25+ T cells represent a unique population of suppressor T cells that can prevent both the initiation of organ-specific autoimmune disease after day 3 thymectomy and the effector function of cloned autoantigen-specific CD4+ T cells. To analyze the mechanism of action of these cells, we established an in vitro model system that mimics the function of these cells in vivo. Purified CD4+CD25+ cells failed to proliferate after stimulation with interleukin (IL)-2 alone or stimulation through the T cell receptor (TCR). When cocultured with CD4+CD25- cells, the CD4+CD25+ cells markedly suppressed proliferation by specifically inhibiting the production of IL-2. The inhibition was not cytokine mediated, was dependent on cell contact between the regulatory cells and the responders, and required activation of the suppressors via the TCR. Inhibition could be overcome by the addition to the cultures of IL-2 or anti-CD28, suggesting that the CD4+CD25+ cells may function by blocking the delivery of a costimulatory signal. Induction of CD25 expression on CD25- T cells in vitro or in vivo did not result in the generation of suppressor activity. Collectively, these data support the concept that the CD4+CD25+ T cells in normal mice may represent a distinct lineage of "professional" suppressor cells.

Figures

Figure 1
Figure 1
Characterization of CD4+CD25+ cells. (A) Total lymph node cells (left) or purified CD4+CD25+ cells (right) from BALB/c mice were stained with PE-conjugated anti-CD4 and biotin-conjugated anti-CD25 followed by FITC-conjugated streptavidin. (B) Purified CD4+ CD25− cells or purified CD4+CD25+ cells were double stained for CD25 and the indicated surface molecules. (C) Proliferative responses of CD4+ CD25+ cells. Cells (5 × 104) were incubated with the indicated agents (10 ng/ml PMA, 1 mM ionomycin, 3 ng/ml IL-2, 3.0 μg/ml soluble anti-CD3, 3.0 μg/ml Con A, 10 μg/ml anti-CD28, or wells coated with 10 μg/ml anti-CD3) in the presence of AC (5 × 104). Results are expressed as the mean of triplicate cultures.
Figure 2
Figure 2
CD4+CD25+ cells suppress the proliferation of CD4+ cells. (A) CD4+CD25− cells (5 × 104) were incubated with plate bound anti-CD3 (triangles) or with 1.0 μg/ml soluble anti-CD3 (squares) in the presence of AC (5 × 104) and the indicated number of CD4+CD25+ cells. (B) CD4+CD25+ cells do not suppress antigen-specific CD4+ cells from TCR transgenic mice. CD4+CD25− cells (5 × 104) were purified from DO.11.10 SCID mice on a BALB/c background and stimulated with 0.5 μM ovalbumin peptide (amino acid 323–339; triangles) or 0.5 μg/ml anti-CD3 (squares) in the presence of AC (5 × 104) and the indicated number of CD4+CD25+ cells. Results are expressed as the mean of triplicate cultures.
Figure 3
Figure 3
PCR analysis of CD4+CD25+ cells. RNA was purified from 6–8 × 106 CD4+CD25− or CD4+CD25+ cells either unstimulated or stimulated with an equivalent number of AC and 0.5 μg/ml anti-CD3 for 15 h. RNA was reverse transcribed and primers for the indicated genes were used to amplify the cDNA. The number of cycles for each primer set are as follows: β-actin, 20 cycles; IL-2, 25 cycles; IL-4, IL-10, TNF-α, and FasL, 30 cycles.
Figure 4
Figure 4
A soluble factor does not mediate CD4+CD25+ induced suppression. (A) CD4+CD25− cells (5 × 105) were cultured in 24-well plates in the presence of 3.0 μg/ml soluble anti-CD3 and AC (5 × 105). The indicated number of CD4+CD25+ cells was added directly to the culture (squares) or to the transwell in the absence (triangles) or presence (circles) of AC (5 × 105). (B) CD4+CD25− cells (5 × 104) were cultured with AC (5 × 104), 0.5 μg/ ml anti-CD3, and 10 μg/ml of the indicated antibodies in the absence (white bars) or presence (black bars) of CD4+ CD25+ cells (2.5 × 104). (C) Supernatants were collected after a 48-h stimulation with soluble anti-CD3 and AC (5 × 104) from CD4+CD25− cells alone (5 × 104), CD4+CD25+ cells alone (5 × 104), or CD4+CD25− (5 × 104) cells coincubated with CD4+CD25+ cells (2.5 × 104). Supernatants (0.1 ml) were then added to CD4+ cells (5 × 104) stimulated with anti-CD3 and AC (5 × 104). Results are expressed as the mean of triplicate cultures.
Figure 4
Figure 4
A soluble factor does not mediate CD4+CD25+ induced suppression. (A) CD4+CD25− cells (5 × 105) were cultured in 24-well plates in the presence of 3.0 μg/ml soluble anti-CD3 and AC (5 × 105). The indicated number of CD4+CD25+ cells was added directly to the culture (squares) or to the transwell in the absence (triangles) or presence (circles) of AC (5 × 105). (B) CD4+CD25− cells (5 × 104) were cultured with AC (5 × 104), 0.5 μg/ ml anti-CD3, and 10 μg/ml of the indicated antibodies in the absence (white bars) or presence (black bars) of CD4+ CD25+ cells (2.5 × 104). (C) Supernatants were collected after a 48-h stimulation with soluble anti-CD3 and AC (5 × 104) from CD4+CD25− cells alone (5 × 104), CD4+CD25+ cells alone (5 × 104), or CD4+CD25− (5 × 104) cells coincubated with CD4+CD25+ cells (2.5 × 104). Supernatants (0.1 ml) were then added to CD4+ cells (5 × 104) stimulated with anti-CD3 and AC (5 × 104). Results are expressed as the mean of triplicate cultures.
Figure 4
Figure 4
A soluble factor does not mediate CD4+CD25+ induced suppression. (A) CD4+CD25− cells (5 × 105) were cultured in 24-well plates in the presence of 3.0 μg/ml soluble anti-CD3 and AC (5 × 105). The indicated number of CD4+CD25+ cells was added directly to the culture (squares) or to the transwell in the absence (triangles) or presence (circles) of AC (5 × 105). (B) CD4+CD25− cells (5 × 104) were cultured with AC (5 × 104), 0.5 μg/ ml anti-CD3, and 10 μg/ml of the indicated antibodies in the absence (white bars) or presence (black bars) of CD4+ CD25+ cells (2.5 × 104). (C) Supernatants were collected after a 48-h stimulation with soluble anti-CD3 and AC (5 × 104) from CD4+CD25− cells alone (5 × 104), CD4+CD25+ cells alone (5 × 104), or CD4+CD25− (5 × 104) cells coincubated with CD4+CD25+ cells (2.5 × 104). Supernatants (0.1 ml) were then added to CD4+ cells (5 × 104) stimulated with anti-CD3 and AC (5 × 104). Results are expressed as the mean of triplicate cultures.
Figure 5
Figure 5
CD4+CD25+ cells from IL-10−/− and IL-4−/− mice suppress proliferation. CD4+CD25+ cells were purified from (A) IL-4−/− and BALB/c control mice or (B) IL-10−/− and C57BL/10 mice, and the indicated numbers were coincubated with CD4+CD25− cells (5 × 104) from control mice stimulated with 0.5 μg/ml anti-CD3 and AC (5 × 104). Results are expressed as the mean of triplicate cultures.
Figure 6
Figure 6
Abrogation of suppression by IL-2 or anti-CD28. CD4+ CD25− cells (5 × 104) were cultured in the presence or absence of CD4+CD25+ cells (2.5 × 104) and 10 μg/ml of the indicated antibodies or 3 ng/ml IL-2. Results are expressed as the mean of triplicate cultures.
Figure 7
Figure 7
CD4+CD25+ cells suppress IL-2 production. (A) CD4+ CD25− cells (5 × 104), AC (5 × 104), and 0.5 μg/ml anti-CD3 were cultured in the presence or absence of CD4+CD25+ cells (2.5 × 104), supernatants were taken at the indicated times and IL-2 was quantified. Each sample was tested in triplicate. (B) RNA was purified from CD4+ cells stimulated in the presence or absence of CD4+CD25+ cells after 16 h using RNAzol B (Tel-test). RNA was separated by gel electrophoresis, transferred to nitrocellulose and probed for IL-2 message (top) or β-actin message (bottom) by using an IL-2 PCR fragment or a β-actin PCR product as a probe.
Figure 8
Figure 8
(A) CD4+CD25+ cells were purified from BALB/c mice while CD4+CD25− cells were obtained by cell sorting and were cultured with AC and Con A for 24 h and were then used as induced CD4+CD25+ cells. Induced or control CD4+CD25+ cells were then coincubated with 0.5 μg/ml anti-CD3 and AC. (B) CD4+CD25+ cells were purified from 3dTx mice or BALB/c control mice and coincubated with CD4+CD25− cells from control mice stimulated with 0.5 μg/ml anti-CD3 and AC. Results are expressed as the mean of triplicate cultures.
Figure 8
Figure 8
(A) CD4+CD25+ cells were purified from BALB/c mice while CD4+CD25− cells were obtained by cell sorting and were cultured with AC and Con A for 24 h and were then used as induced CD4+CD25+ cells. Induced or control CD4+CD25+ cells were then coincubated with 0.5 μg/ml anti-CD3 and AC. (B) CD4+CD25+ cells were purified from 3dTx mice or BALB/c control mice and coincubated with CD4+CD25− cells from control mice stimulated with 0.5 μg/ml anti-CD3 and AC. Results are expressed as the mean of triplicate cultures.

References

    1. Schwartz RH. A cell culture model for T lymphocyte clonal anergy. Science. 1990;248:1349–1356.
    1. Miller JFAP, Heath WR. Self-ignorance in the peripheral T-cell pool. Immunol Rev. 1993;133:131–150.
    1. Wucherpfennig KW, Strominger JL. Molecular mimicry in T cell–mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell. 1995;80:695–705.
    1. Möller, G., editor. 1996. Dominant immunological tolerance. Immunol. Rev. 149:1–243.
    1. Fowell D, Mason D. Evidence that the T cell repertoire of normal rats contains cells with the potential to cause diabetes. Characterization of the CD4+T cell subset that inhibits this autoimmune potential. J Exp Med. 1993;177:627–636.
    1. Saoudi A, Seddon B, Heath V, Fowell D, Mason D. The physiological role of regulatory T cells in the prevention of autoimmunity: the function of the thymus in the generation of the regulatory T cell subset. Immunol Rev. 1996;149:195–216.
    1. Powrie F, Leach MW, Mauze S, Caddle LB, Coffman RL. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C.B-17 scid mice. Int Immunol. 1993;51:1461–1471.
    1. Mordes JP, Gallina DL, Handler ES, Greiner DL, Nakamura N, Pelletier A, Rossini AA. Transfusions enriched for W3/25+ helper/inducer T lymphocytes prevent spontaneous diabetes in the BB/W rat. Diabetologia. 1987;30:22–26.
    1. Shimada A, Charlton B, Rohane P, Taylor-Edwards C, Fathman CG. Immune regulation in type 1 diabetes. J Autoimmun. 1996;9:263–269.
    1. Lafaille JJ, Nagashima K, Katsuki M, Tonegawa S. High incidence of spontaneous autoimmune encephalomyelitis in immunodeficient anti-myelin basic protein T cell receptor transgenic mice. Cell. 1994;78:399–408.
    1. Kojima A, Prehn RT. Genetic susceptibility to post-thymectomy autoimmune diseases in mice. Immunogenetics. 1981;14:15–27.
    1. Sakaguchi S, Fukuma K, Kuribayashi K, Masuda T. Organ-specific autoimmune diseases induced in mice by elimination of T cell subset. I. Evidence for the active participation of T cells in natural self-tolerance; deficit of a T cell subset as a possible cause of autoimmune disease. J Exp Med. 1985;161:72–87.
    1. Asano M, Toda M, Sakaguchi N, Sakaguchi S. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J Exp Med. 1996;184:387–396.
    1. Suri-Payer E, Kehn PJ, Cheever AW, Shevach EM. Pathogenesis of post-thymectomy autoimmune gastritis. Identification of anti-H/K adenosine triphosphatase-reactive T cells. J Immunol. 1996;157:1799–1805.
    1. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995;155:1151–1164.
    1. Suri-Payer E, Amar AZ, Thornton AM, Shevach EM. CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J Immunol. 1998;160:1212–1218.
    1. Noben-Trauth N, Köhler G, Burki K, Ledermann B. Efficient targeting of the IL-4 gene in a BALB/c embryonic stem cell line. Transgenic Res. 1996;5:487–491.
    1. Bonomo A, Kehn PJ, Payer E, Rizzo L, Cheever AW, Shevach EM. Pathogenesis of post-thymectomy autoimmunity. Role of syngeneic MLR-reactive T cells. J Immunol. 1995;154:6602–6611.
    1. Segal BM, Shevach EM. IL-12 unmasks latent autoimmune disease in resistant mice. J Exp Med. 1996;184:771–775.
    1. Walunas TL, Lenschow DJ, Bakker CY, Linsley PS, Freeman GJ, Green JM, Thompson CB, Bluestone JA. CTLA-4 can function as a negative regulator of T cell activation. Immunity. 1994;1:405–413.
    1. Benacerraf B, Germain RN. A single major pathway of T-lymphocyte interactions in antigen-specific immune suppression. Scand J Immunol. 1981;13:1–10.
    1. Groux H, O'Garra A, Bigler M, Rouleau M, Antonenko S, de Vries JE, Roncarolo MG. A CD4+T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature. 1997;389:737–742.
    1. Kulkarni AB, Huh CG, Becker D, Geiser A, Lyght M, Flanders KC, Roberts AB, Sporn MB, Ward JM, Karlsson S. Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci USA. 1993;90:770–774.
    1. Powrie F, Carlino J, Leach MW, Mauze S, Coffman RL. A critical role for transforming growth factor-β but not interleukin 4 in the suppression of T helper type 1–mediated colitis by CD45RBlow CD4+T cells. J Exp Med. 1996;183:2669–2674.
    1. Chen Y, Inobe J, Kuchroo VK, Baron JL, Janeway CA, Jr, Weiner HL. Oral tolerance in myelin basic protein T-cell receptor transgenic mice: suppression of autoimmune encephalomyelitis and dose-dependent induction of regulatory cells. Proc Natl Acad Sci USA. 1996;93:388–391.
    1. Han HS, Jun HS, Utsugi T, Yoon JW. Molecular role of TGF-beta, secreted from a new type of CD4+ suppressor T cell, NY4.2, in the prevention of autoimmune IDDM in NOD mice. J Autoimmun. 1997;10:299–307.
    1. Field EH, Ye ZX, Rouse TM. Mechanisms of tolerance induction: splenocytes from total lymphoid irradiated mice inhibit the IL-2 pathway in TCR-activated CD4 cells. J Immunol. 1996;157:3796–3803.
    1. Krämer S, Schimpl A, Hünig T. Immunopathology of interleukin (IL) 2–deficient mice: thymus dependence and suppression by thymus-dependent cells with an intact IL-2 gene. J Exp Med. 1995;182:1769–1776.
    1. Sadlack B, Löhler J, Schorle H, Klebb G, Haber H, Sickel E, Noelle RJ, Horak I. Generalized autoimmune disease in interleukin-2–deficient mice is triggered by an uncontrolled activation and proliferation of CD4+ T cells. Eur J Immunol. 1995;25:3053–3059.
    1. Willerford DM, Chen J, Ferry JA, Davidson L, Ma A, Alt FW. Interleukin-2 receptor α chain regulates the size and content of the peripheral lymphoid compartment. Immunity. 1995;3:521–530.
    1. Suzuki H, Kündig TM, Furlonger C, Wakeham A, Timms E, Matsuyama T, Schmits R, Simard JJL, Ohashi PS, Griesser H, et al. Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor β. Science. 1995;268:1472–1476.
    1. Sakaguchi S, Toda M, Asano M, Itoh M, Morse SS, Sakaguchi N. T cell–mediated maintenance of natural self-tolerance: its breakdown as a possible cause of various autoimmune diseases. J Autoimmun. 1996;9:211–220.
    1. Taguchi O, Nishizuka Y. Self tolerance and localized autoimmunity. Mouse models of autoimmune disease that suggest tissue-specific suppressor T cells are involved in self tolerance. J Exp Med. 1987;165:146–156.
    1. Taguchi O, Kontani K, Ikeda H, Kezuka T, Takeuchi M, Takahashi T. Tissue-specific suppressor T cells involved in self-tolerance are activated extrathymically by self-antigens. Immunology. 1994;89:13–19.
    1. Smith H, Sakamoto Y, Kasai K, Tung KS. Effector and regulatory cells in autoimmune oophoritis elicited by neonatal thymectomy. J Immunol. 1991;147:2928–2933.
    1. Saoudi A, Seddon B, Fowell D, Mason D. The thymus contains a high frequency of cells that prevent autoimmune diabetes on transfer into prediabetic recipients. J Exp Med. 1996;184:2393–2398.
    1. Kumar V, Aziz F, Sercarz E, Miller A. Regulatory T cells specific for the same framework 3 region of the Vβ8.2 chain are involved in the control of collagen II–induced arthritis and experimental autoimmune encephalomyelitis. J Exp Med. 1997;185:1725–1733.
    1. Kumar V, Sercarz E. T cell regulatory circuitry: antigen-specific and TCR-idiopeptide–specific T cell interactions in EAE. Int Rev Immunol. 1993;9:287–297.
    1. Lombardi G, Sidhu S, Batchelor R, Lechler R. Anergic T cells as suppressor cells in vitro. Science. 1994;264:1587–1589.
    1. Cobbold SP, Adams E, Marshall SE, Davies JD, Waldman H. Mechanisms of peripheral tolerance and suppression induced by monoclonal antibodies to CD4 and CD8. Immunol Rev. 1996;149:5–33.

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